Invasion of tumor cells through basement membranes and interstitial tissues allows tumors to metastasize and colonize new sites in the body. However, the manner in which cell invasion is regulated is completely unknown. Recent studies indicate that tyrosine phosphorylation of membrane-bound proteins is critically important in metastatic invasion of cells. These studies show that tyrosine kinases and their membrane-bound substrates are highly elevated in association with invadopodia, the cellular protrusions at the leading edge of invasive cells. Formation of invadopodia and subsequent cell invasion were blocked by genistein, an inhibitor of tyrosine kinases. Invadopodia and shed membrane vesicles of the human malignant melanoma cell line LOX, as well as membrane vesicles derived from LOX grown in experimental models are a rich source of invasion-related antigens. Starting material for antigen preparation is already available to isolate invasion related-tyrosine phosphorylated proteins and several tyrosine phosphorylated proteins have been identified in isolated invadopodia membrane fractions using existing antibodies. This proposed work will: 1) isolate and microsequence the major unidentified tyrosine phosphorylated polypeptides from invadopodia and shed membrane vesicles of LOX cells, and membranes of LOX tumors using anti- phosphotyrosine monoclonal antibodies, and 2) identify existing and generate new monoclonal antibodies against these invasion-associated tyrosine phosphorylated proteins. Protein sequencing and monoclonal antibodies obtained here will allow us to identity tyrosine phosphorylated proteins that localize exclusively to invasive ruffles and whose association with invadopodia may form the molecular basis for the control of tumor cell invasion and metastasis.